1. Field of the Invention
This invention relates to processes that copy oligonucleotides in a polymerase chain reaction (PCR) where these oligonucleotides incorporate nucleotide analogs (“non-standard nucleotides”) that form base pairs joined by hydrogen bonding patterns not found in standard nucleotides A, T, G and C. The invention relates more specifically to processes that amplify oligonucleotides holding more than one non-standard nucleotides, including non-standard nucleotides at adjacent positions in the oligonucleotides chain, and amplification in a nested PCR format.
2. Description of Related Art
Natural oligonucleotides bind to complementary oligonucleotides according to well-known rules of nucleobase pairing first elaborated by Watson and Crick, where adenine (A) pairs with thymine (T) (or uracil, U, in RNA), and guanine (G) pairs with cytosine (C), with anti-parallel complementary strands. In this disclosure, “DNA”, “oligonucleotide”, or “nucleic acid” is understood to include DNA and RNA, as well as derivatives where the sugar is modified, as in 2′-O-methyl and 2′,3′-dideoxynucleoside derivatives, where the nucleobase has an appendage, and these nucleic acids and their analogs in non-linear topologies, including as dendrimers, comb-structures, and nanostructures, and analogs carrying appendages or tags (e.g., fluorescent, functionalized, or binding, such as biotin). Further, “polymerase” in this application is meant to include DNA polymerases of all families, RNA polymerases, and reverse transcriptases.
These pairing rules allow specific hybridization of oligonucleotides to complementary oligonucleotides, making oligonucleotides valuable as probes in the laboratory, in diagnostics, as messages that direct the synthesis of proteins, and in other applications known in the art. Such pairing is used, for example and without limitation, to capture oligonucleotides to beads, arrays, and other solid supports, allow nucleic acids to fold in hairpins, beacons, and catalysts, support function, such as fluorescence, quenching, binding/capture, and catalysis, and as part of complex structures, including dendrimers and nanostructures, and scaffolds to guide chemical reactions.
Further, base pairing underlies the enzymatic synthesis of oligonucleotides complementary to a template. Here, assembly of building blocks from nucleoside triphosphates is directed by a template to form a complementary oligonucleotide with a complementary sequence. This is the basis for replication in living systems, and underlies technologies for enzymatic synthesis and amplification of specific nucleic acids by enzymes such as DNA and RNA polymerase, the polymerase chain reaction (PCR), and assays involving synthesis, ligation, cleavage, immobilization and release, inter alia.
Watson-Crick pairing rules can be understood as the product of two rules of complementarity: (1) size complementarity (a big purine pairs with a small pyrimidine) and (2) hydrogen bonding complementarity (hydrogen bond donors pair with hydrogen bond acceptors). However, as noted by U.S. Pat. Nos. 5,432,272, 5,965,364, 6,001,983, 6,037,120, 6,140,496, 6,627,456, and 6,617,106, Watson-Crick geometry can accommodate as many as 12 nucleobases forming 6 mutually exclusive pairs. Of these, four nucleobases forming two pairs are designated “standard”, while eight nucleobases forming four pairs were termed “non-standard”, and may be part of an “artificially expanded genetic information system” (AEGIS).
To systematize the nomenclature for the hydrogen bonding patterns, the hydrogen bonding pattern implemented on a small component of a nucleobase pair are designated by the prefix “py”. Following this prefix is the order, from the major to the minor groove, of hydrogen bond acceptor (A) and donor (D) groups. Thus, both thymine and uracil implement the standard hydrogen bonding pattern pyADA. The standard nucleobase cytosine implements the standard hydrogen bonding pattern pyDAA. Hydrogen bonding patterns implemented on the large component of the nucleobase pair are designated by the prefix “pu”. Following the prefix, hydrogen bond donor and acceptor groups are designated, from major to minor groove, by “A” and “D”. Thus, the standard nucleobases adenine and guanine implement the standard hydrogen bonding patterns puDA- and puADD respectively.
A central teaching of this disclosure is that hydrogen-bonding patterns are distinct from the organic molecule that implemented them. Thus, guanosine implements the puADD hydrogen-bonding pattern. So does, however, 7-deazaguanosine, 3,7-dideazaguanosine, and many other purines and purine analogs, including those that carry side chains carrying functional groups, such as biotin, fluorescent, and quencher groups. Which organic molecule is chosen to implement a specific hydrogen-bonding pattern determines, in part, the utility of the non-standard hydrogen-bonding pattern, in various applications to which it might be applied.
Claims of U.S. Pat. No. 5,432,272 and its successors covered non-standard bases that implemented the pyDDA hydrogen bonding pattern. Subsequent efforts to use these, however, encountered problems, including epimerization [Voe96a,b], oxidation [Von95], and uncharacterized decomposition. Accordingly, Benner invented a new non-standard nucleoside, 6-amino-5-nitro-3-(1′-beta-D-2′-deoxyribofuranosyl)-2-(1H)-pyridone (dZ) to implement the pyDDA hydrogen bonding pattern. The nitro group rendered the otherwise electron-rich heterocycle stable against both oxidation and epimerization under standard conditions. When paired with a corresponding puAAD nucleotide, duplexes were formed with stabilities that, in many cases, were higher than those observed in comparable strands incorporating the dG:dC nucleobase pair [Yan06]. This invention is covered by U.S. patent application Ser. No. 11/372,400, which is incorporated herein by reference. Contents of this patent application have been published [Hut03].
While Z supports binding of oligonucleotides containing it to complementary strands that match a nucleobase implementing the puAAD hydrogen bond pattern, it was not clear that polymerases would accept this unnatural base pair. Polymerases are known to be idiosyncratic [Hor95], meaning that experimentation is necessary to ascertain whether a specific implementation of a non-standard hydrogen bonding scheme can be accepted by a polymerase.
Thus, it was necessary to show by experiment that polymerases could incorporate dZ and dP. This was done for oligonucleotides containing a single dZ or dP [Yan07], which was published less than a year before the priority date of the instant application. However, [Yan07] showed that the dZ and dP are lost in multiple PCR cycles with Taq and Deep Vent (exo−) polymerases, perhaps via a mechanism where deprotonated dZ mispairs with dG (or deprotonated dG pairs with dZ), while protonated dC mispairs with dP (or protonated dP pairs with dC). Thus, this art teaches away from any use of the non-standard dZ:dP nucleobase pair in higher level PCR, defined as PCR that creates amplicons with multiple non-standard nucleotides.